Sculpting nuclear envelope identity from the endoplasmic reticulum during the cell cycle.

The nuclear envelope (NE) regulates nuclear functions, including transcription, nucleocytoplasmic transport, and protein quality control. While the outer membrane of the NE is directly continuous with the endoplasmic reticulum (ER), the NE has an overall distinct protein composition from the ER, which is crucial for its functions. During open mitosis in higher eukaryotes, the NE disassembles during mitotic entry and then reforms as a functional territory at the end of mitosis to reestablish nucleocytoplasmic compartmentalization. In this review, we examine the known mechanisms by which the functional NE reconstitutes from the mitotic ER in the continuous ER-NE endomembrane system during open mitosis. Furthermore, based on recent findings indicating that the NE possesses unique lipid metabolism and quality control mechanisms distinct from those of the ER, we explore the maintenance of NE identity and homeostasis during interphase. We also highlight the potential significance of membrane junctions between the ER and NE.

Eisosome protein Pil1 regulates mitochondrial morphology, mitophagy, and cell death in Saccharomyces cerevisiae.

Mitochondrial morphology and dynamics maintain mitochondrial integrity by regulating its size, shape, distribution, and connectivity, thereby modulating various cellular processes. Several studies have established a functional link between mitochondrial dynamics, mitophagy, and cell death, but further investigation is needed to identify specific proteins involved in mitochondrial dynamics. Any alteration in the integrity of mitochondria has severe ramifications that include disorders like cancer and neurodegeneration. In this study, we used budding yeast as a model organism and found that Pil1, the major component of the eisosome complex, also localizes to the periphery of mitochondria. Interestingly, the absence of Pil1 causes the branched tubular morphology of mitochondria to be abnormally fused or aggregated, whereas its overexpression leads to mitochondrial fragmentation. Most importantly, pil1Δ cells are defective in mitophagy and bulk autophagy, resulting in elevated levels of reactive oxygen species and protein aggregates. In addition, we show that pil1Δ cells are more prone to cell death. Yeast two-hybrid analysis and co-immunoprecipitations show the interaction of Pil1 with two major proteins in mitochondrial fission, Fis1 and Dnm1. Additionally, our data suggest that the role of Pil1 in maintaining mitochondrial shape is dependent on Fis1 and Dnm1, but it functions independently in mitophagy and cell death pathways. Together, our data suggest that Pil1, an eisosome protein, is a novel regulator of mitochondrial morphology, mitophagy, and cell death.

An adaptable live-cell imaging protocol to analyze organelle morphology in Saccharomyces cerevisiae.

The protocol describes semiautomated live cell imaging in budding yeast. A key feature of the protocol is immobilizing cells in a culture dish, which allows for longer imaging times, changing culture media, or drug treatments. We describe steps for image acquisition and deconvolution, followed by manual analysis of quantifiable parameters to represent morphological changes in nuclear shape. We compare wild type with ssf1Δ, which is known to alter nuclear morphology. The protocol can be adapted to other organelles and processes. For complete details on the use and execution of this profile, please refer to Male et al., 2020, Deolal et al. (2021).

Uip4p modulates nuclear pore complex function in Saccharomyces cerevisiae.

A double membrane bilayer perforated by nuclear pore complexes (NPCs) governs the shape of the nucleus, the prominent distinguishing organelle of a eukaryotic cell. Despite the absence of lamins in yeasts, the nuclear morphology is stably maintained and shape changes occur in a regulated fashion. In a quest to identify factors that contribute to regulation of nuclear shape and function in Saccharomyces cerevisiae, we used a fluorescence imaging based approach. Here we report the identification of a novel protein, Uip4p, that is required for regulation of nuclear morphology. Loss of Uip4 compromises NPC function and loss of nuclear envelope (NE) integrity. Our localization studies show that Uip4 localizes to the NE and endoplasmic reticulum (ER) network. Furthermore, we demonstrate that the localization and expression of Uip4 is regulated during growth, which is crucial for NPC distribution.

Regulation of diverse nuclear shapes: pathways working independently, together.

Membrane-bound organelles provide physical and functional compartmentalization of biological processes in eukaryotic cells. The characteristic shape and internal organization of these organelles is determined by a combination of multiple internal and external factors. The maintenance of the shape of nucleus, which houses the genetic material within a double membrane bilayer, is crucial for a seamless spatio-temporal control over nuclear and cellular functions. Dynamic morphological changes in the shape of nucleus facilitate various biological processes. Chromatin packaging, nuclear and cytosolic protein organization, and nuclear membrane lipid homeostasis are critical determinants of overall nuclear morphology. As such, a multitude of molecular players and pathways act together to regulate the nuclear shape. Here, we review the known mechanisms governing nuclear shape in various unicellular and multicellular organisms, including the non-spherical nuclei and non-lamin-related structural determinants. The review also touches upon cellular consequences of aberrant nuclear morphologies.

The challenge of staying in shape: nuclear size matters.

Cellular organelles have unique morphology and the organelle size to cell size ratio is regulated. Nucleus is one of the most prominent, usually round in shape, organelle of a eukaryotic cell that occupies 8-10% of cellular volume. The shape and size of nucleus is known to undergo remodeling during processes such as cell growth, division and certain stresses. Regulation of protein and lipid distribution at the nuclear envelope is crucial for preserving the nuclear morphology and size. As size and morphology are interlinked, altering one influences the other. In this perspective, we discuss the relationship between size and shape regulation of the nucleus.

Nucleolar size regulates nuclear envelope shape in Saccharomyces cerevisiae.

Nuclear shape and size are cell-type specific. Change in nuclear shape is seen during cell division, development and pathology. The nucleus of Saccharomycescerevisiae is spherical in interphase and becomes dumbbell shaped during mitotic division to facilitate the transfer of one nucleus to the daughter cell. Because yeast cells undergo closed mitosis, the nuclear envelope remains intact throughout the cell cycle. The pathways that regulate nuclear shape are not well characterized. The nucleus is organized into various subcompartments, with the nucleolus being the most prominent. We have conducted a candidate-based genetic screen for nuclear shape abnormalities in S. cerevisiae to ask whether the nucleolus influences nuclear shape. We find that increasing nucleolar volume triggers a non-isometric nuclear envelope expansion resulting in an abnormal nuclear envelope shape. We further show that the tethering of rDNA to the nuclear envelope is required for the appearance of these extensions.

Impaired neuronal and astroglial metabolic activity in chronic unpredictable mild stress model of depression: Reversal of behavioral and metabolic deficit with lanicemine.

Major depressive disorder is the leading cause of disability and suicidality worldwide. Here, we evaluated neural metabolic activity in prefrontal cortex (PFC) in C57BL6 mice undergoing a chronic unpredictable mild stress (CUMS) for three weeks to induce depression. Further, the efficacy of Lanicemine, a low trapping NMDA receptor antagonist, on behavioral and neurometabolic measures in CUMS mice was evaluated. The PFC neuronal and astroglial metabolic activity was evaluated by Proton Observed Carbon Edited (POCE) MR spectroscopy together with an infusion of [1,6-13C2]glucose and [2-13C]acetate, respectively. The rates of glutamatergic, GABAergic and astrocytic TCA cycles and neurotransmitter cycling were obtained by fitting a three-compartment metabolic model to 13C turnover of amino acids. Mice subjected to CUMS exhibited significantly reduced sucrose preference (CUMS 58.0 ± 12.5%, n = 29; Control 86.3 ± 6.4%, n = 30; p < 0.0001), and increased immobility (CUMS 146.1 ± 60.8s, n = 29; Control 29.9 ± 19.3s, n = 30; p < 0.0001) in the forced swim test. The concentrations of 13C labeled amino acids from [2-13C]acetate were decreased suggesting reduced astroglial metabolic activity in CUMS mice. The glutamatergic and GABAergic TCA cycle rates were decreased in CUMS mice when compared with controls. In addition, GABA-glutamine and glutamate-glutamine neurotransmitter cycling were reduced in mice subjected to CUMS regimen. Most interestingly, a short time intervention of lanicemine restored behavioral measures (sucrose preference and immobility), and rates of glucose oxidation in glutamatergic and GABAergic neurons in CUMS mice. In summary, our findings suggest that depression leads to a reduction in excitatory and inhibitory neurotransmission in PFC, and targeting glutamatergic pathway may have potential therapeutic role in chronic depression.